1. Introduction
Human exposure to aluminum (Al) is unavoidable during the entire lifespan, and the numerous ways we are exposed to Al ensures that it will be notably more so in the future [
1]. However, the mechanisms of absorption, metabolism, deposition, and excretion of Al through the human body still need to be well elucidated in part due to the absence of biological function of this non-essential metal [
2]. Furthermore, the consequences for human health from exposure to Al continue to be reported in the scientific literature [
3,
4], but they are still mostly unknown compared to other human contaminants [
5,
6].
Human exposure to Al occurs through dietary and non-dietary sources. Al salts are added to commercially available foods and are used to treat drinking water and the packaging and storage of various food products [
7]. Among the non-dietary sources, Al is found in pharmaceuticals, cosmetics, and deodorants and is used as an adjuvant to improve vaccines’ immune response [
8]. Upon reaching a body burden threshold, Al shows a non-linear dose-response toxicity, as evidenced by recent research [
9,
10].
Al exposure triggers hematological, renal, cardiovascular, endocrine, reproductive, and auto-immune disorders [
11,
12,
13,
14]. Al
+3 appears to disproportionally increase reactive oxygen production through Fenton reaction and trigger immune responses which may contribute to chronic systemic inflammation [
14,
15]. Al has been considered a trigger of the onset, progression, and outcomes of Alzheimer’s disease (AD) [
16,
17,
18]. Although the relationship to metal exposure has been documented to be strongly related to damage to the nervous system and associated with neurodegenerative diseases, it seems not restricted to the central nervous system.
An elevated concentration of Al has been found in human sperm in correlation with a reduction in sperm quality [
13]. Experimentally, rodents exposed to considerably high Al levels develop testicular abnormalities, endocrine disruption, and impaired reproduction. Based on this, the effects of burgeoning human Al exposure on female and male reproductive systems have been gaining attention during the last decades, mainly due to the increased infertility rate among couples and the exponential decrease in human sperm quality [
19,
20]. Recently, we have investigated the effects of Al exposure at human dietary levels, and we observed that it could impair spermatogenesis and sperm quality [
21]. Moreover, it has been described that reproductive dysfunction can be achieved even at low doses of Al [
21,
22].
Considering the growing and inevitable human exposure to Al, strategies are necessary to counteract or reduce its impact on human and reproductive health. Our group has tested the bioactive properties of an egg white hydrolysate (EWH) treated for 8 h with pepsin against numerous cardiometabolic complications and metal exposure consequences [
23,
24,
25]. These multi-functional peptides have potent anti-inflammatory and antioxidant capacities [
23,
24,
25], which could be of value in preventing or minimizing the consequences of Al exposure. In addition, in a model of Al exposure at human dietary levels, EWH prevented memory impairment and vascular dysfunction after long-term exposure [
26,
27].
Here, we report our investigation of the anti-inflammatory and antioxidant regulatory effects of EWH as protection against reproductive dysfunction induced by low and high levels of dietary Al in Wistar rats. This study highlights for the first time that dietary supplementation with EWH peptides protects against reduced sperm quality after Al exposure at low and high dietary Al levels.
4. Discussion
Results of our study highlight the capacity of dietary supplementation with bioactive peptides from an antioxidant and anti-inflammatory EWH to prevent or reduce the deleterious effects of Al exposure on the male reproductive system. The harmful effects of Al exposure have been demonstrated to be equivalent to low or high human dietary levels on the reproductive system, but not following a logical dose-response behavior. Some complications associated with Al exposure are not dependent on the concentration of the metal, but rather on the stimulus (pathways) that this metal could be triggering. In previous works, Al accumulated in testis induced macrophage activation and increased RS production in reproductive organs. These actions impair sperm characteristics needed for its viability, such as progressive motility and normal morphology, and sperm production in the testis with consequent sperm count reduction in the testis and epididymis of male rats [
21].
We previously reported the in vitro antioxidant properties of EWH [
24,
39]. After in vivo studies, we determined that the consumption of EWH prevented inflammation and oxidative stress in different models of cardiometabolic disease and metal exposures [
25,
29,
40]. In the present study, the oral uptake of 1 g/kg b.w. of pepsin-EWH prevented the reproductive dysfunction induced by dietary Al exposure at low and high intakes. In addition, the EWH prevented Al deposition within the testis, controlled inflammation, oxidative stress, and impaired spermatogenesis and sperm quality observed after Al exposure.
Exposure to Al in the general population, including susceptible groups such as children or pregnant women, is continuously increasing [
41,
42]. However, very few studies have addressed the consequences of Al exposure on human health. Our study supports the hypothesis that less is not precisely safer in the case of Al exposure and that small amounts of Al are sufficient to trigger adverse effects. We know that animal model findings cannot be directly extrapolated to human exposures; however, outcomes due to the constancy observed in the results obtained with different animal models of Al exposure [
9,
43] raise a concern about exposure to Al in the general population, which could have worse consequences in a more susceptible population. Supporting this concern, a recent metallome study in pregnant women revealed that Al might play a role in the onset of central nervous system malformations. Specifically, a direct relationship was found between maternal serum concentration of Al and congenital defects of the central nervous system [
44].
In the global scenario of declining sperm quality and increasing male-female couples suffering from infertility, it is essential to understand the causalities and contributors to this phenomenon [
20,
45]. Male factors account for 50% of worldwide infertility cases, and accumulating evidence supports the link between exposure to contaminants and lifestyles to male reproductive function [
46,
47,
48].
The presence of Al in seminal human plasma has been investigated in the last decades, and a high concentration of Al seems to be related to low sperm quality, specifically reduced motility and sperm count among individuals showing some rate of reproductive dysfunction [
13,
49,
50]. Al exposure in vitro seems to have a higher impact on human sperm motility and MDA production when compared with other common contaminants such as cadmium or lead [
51]. Rodent models of exposure to Al support its impact on male reproductive health [
52,
53]. Al at doses ranging from 25 mg/kg b.w./day to 256.72 mg/kg b.w./day and different routes of exposure seem to disrupt the neuroendocrine system, reducing testosterone, luteinizing and follicular stimulating hormone, act locally in testis and epididymis inducing germ cell degeneration, atrophy and apoptosis and increasing sperm abnormalities in rodents [
53,
54,
55].
The doses of Al of 8.3 mg/kg b.w./day, previously developed by our group, might be considered equivalent to a normal dietary intake of Al by human [
10]. This dose was calculated according to a published protocol described by [
56] and translated to an animal dose, based on a body surface area normalization method [
57]. The high dose of Al exposure at 100 mg/kg b.w./day, is a protocol known to promote cognitive impairment in rats [
30].
Herein, we have found that dietary supplementation with a pepsin-EWH was able to prevent the deleterious effects on reproductive organs after Al exposure at either low or high human-relevant doses. Specifically, in the animal model, we have shown that Al at low or high human dietary level impairs sperm function and spermatogenesis and that a pepsin-EWH added to the animal diet as a functional food ingredient prevented the effects of Al in testis, epididymis, and prostate, maintaining the sperm production per testis and the sperm quality similar to sperm quality found in the control animals. Moreover, EWH supplementation prevented Al deposition in the testis, the increased RS and lipid peroxidation levels, the macrophage differentiation and infiltration. Testes and spermatozoa are very susceptible to oxidative damage due to abundant polyunsaturated fatty acids (PUFAs) in the sperm membrane and their limited antioxidant capacity, which appears to be a standard feature of male infertility [
58,
59]. Our results suggest an ability of EWH to counteract the pro-oxidant effects of Al
3+, which could be up-regulating the antioxidant capacity, then reducing oxidative damage within reproductive tissues.
Indeed, the hydrolysis of the EWH with pepsin for 8 h releases peptides with several bioactive properties [
23]. Among them, antioxidant properties of EWH have been demonstrated in vitro [
23,
29], reaching a peroxyl radical-scavenging activity of 574 μmol Trolox/g protein, which seems to be related to the presence of peptide sequences with Tyr at N-terminal residue [
23]. Furthermore, the antioxidant activity of EWH has been proven in animal models [
24,
25,
26,
27,
60]. Therefore, the antioxidant capacity could be related to its protective and beneficial effects against the harmful consequences of Al exposure on the reproductive system.
Previous studies of our group have suggested the functional effect of EWH to protect against neurological, cardiovascular, and reproductive dysfunctions induced by heavy metals at low and high levels [
25,
26,
27,
60,
61]. The daily consumption of pepsin-EWH also modulates inflammation by decreasing plasma levels of tumor necrosis factor-alpha and cyclooxygenase-2 activation in animals [
24,
27]. In the present study, the EWH also prevented the macrophage activation evoked by Al exposure in testis. Therefore, the protective role of daily EWH supplementation seems to be due to controlled oxidative stress and balanced inflammation status.
Considering the antioxidant properties attributed to EWH and that EWH and Al have been administered separately, it is most likely that chelation occurs after both components are metabolized, during their passage through the digestive tract, or after in a localized manner in one of the target tissues. It has been described that food components, including food proteins, can act as potent metal chelators, thereby reducing metal concentration in blood and other organs [
62]. Moreover, the production of hydrolysates derived from food proteins containing peptides with tyrosine and phenylalanine residues that have chelating capacity has been described [
63]. In addition, some authors have suggested that sulfur-rich compounds, as is the case with some proteins and peptides, could reduce the absorption or reabsorption of toxic metals, and enhance natural detoxification pathways, since the efficiency of a chelating compound is related to its sulfur composition, a molecule that has a high affinity for toxic metals [
64]. In fact, egg white is a food matrix largely composed of sulfur-containing proteins [
65]. Therefore, it is conceivable that the antioxidant peptides contained in the egg white hydrolysate used in this work could act as chelators of the metal, thus reducing its absorption and enhancing its natural excretion pathways.
Another underlying mechanism that could be indirectly involved in metal chelation could be derived from the properties of EWH to increase the antioxidant defense of the organism and specifically the levels of reduced glutathione in liver. One of the best-defined mechanisms on the metal detoxification process by chelation is through reduced glutathione [
66]. This molecule can form complexes with different metals. It has been shown that metals can form stable complexes when combined with the sulfhydryl groups of the reduced glutathione molecule in a 1:2 ratio (metal ion: reduced glutathione) [
67]. In previous studies an increase in the levels of this molecule was observed in EWH-treated animals exposed to a toxic metal [
68], and it is possible that this mechanism may also be related with an increase in chelation capacity.
We must emphasize that humans are exposed to Al daily, which means that Al chronically intoxicates most humans. Contrarily, no drugs or chelators are clinically approved to remove Al from the body [
69]. In this context, combined strategies aiming to increase the removal of Al from the human body and to prevent the effects of the remaining Al could be an alternative to reduce the impact of the overall chronic Al intoxication. Drinking silicon-rich mineral water (30 mg/L, 1 L/ day) appears to facilitate the excretion of Al from the body and improve cognitive function in Alzheimer’s patients [
70]. In the reproductive system, the natural active product found in crucifers, such as broccoli sprouts and sulforaphane, showed the potential to lower AlCl
3-induced testicular toxicity in rats [
71]. Furthermore, several different sources of antioxidant substances such as vitamin C [
72], selenium-rich yeast [
73], Citrullus lanatus seed ethanol extract [
74], tyrosyl (an antioxidant present in olive oil) [
75], and curcumin [
76] show protective effects against reproductive Al toxicity probably due to their antioxidant and antiapoptotic properties. Therefore, our results suggest that oral supplementation with pepsin-EWH at 1 g/kg b.w./day could prevent the harmful effects of Al´s increased human body burden. These data, combined with previous research, are encouraging and highlight the health benefits of dietary supplementation with pepsin-EWH, which could be used as a functional food ingredient and introduced into the human diet to help prevent the deleterious outcomes induced by long-term exposure to Al. However, further studies are necessary to better understand the underlying mechanisms of EWH and its health benefits.